Current Protein and Peptide Science - Volume 12, Issue 3, 2011

Since the pioneering work of Anfinsen in 1973, protein folding was subject to many biophysical studies. However, the protein folding studies show a kind of renaissance nowadays. Since protein aggregation is considered as the main cause of several human diseases, an unprecedented number of scientific reports have been published in the last decade on protein aggregation. In fact, protein aggregates as intracellular inclusion or extracellular deposition have been associated with highly prevalent diseases such as Alzheimer's and Parkinson's disease, for which no cure is available yet. In this scenario, scientists from different areas have converged on protein folding and the old paradigms underwent profound changes. The classical energy landscape funnel that explained the folding pathways for years is unable to explain protein aggregation process and is now replaced by a double funnel energy landscape as proposed by Jahn and Radford in 2005. This new concept helps to understand how a native protein that is structural and thermodynamically stable can reach other conformations with even lower free energy than the native state. At this point, the external variables which may disturb the natural folding equilibrium such as pH, temperature, presence of surface or small molecules acquire relevance. Protein misfolding and aggregation studies have produced a unique space in which researchers from biochemistry and biophysics to biomedicine and pharmacology can efficiently interact. This interdisciplinary complementation is essential in order to obtain enough amount of knowledge necessary to completely understand the protein aggregation process. Of course, the language of one area might sometimes be difficult to be fully understood by the others. This is the challenge we have to deal with. But in the end, it will be extremely positive because the contributions from the different areas of science will be essential. Currently, a lot of information exploiting spectroscopic and computing simulation techniques is available giving information on the aggregation pathways of pathologies-related proteins or peptides. In this way, this CPPS issue is devoted to discuss the contributions of classical as well as novel biophysical techniques, defining the state of the art of the molecular mechanisms of protein aggregation. Hopefully it will provide useful tools to researchers from other fields for analysing the current state of knowledge. Moreover, the influence of new actors involved in altering the protein folding equilibrium such as membranes and glycosaminoglycans, are also considered.

Even though our knowledge of how proteins misfold and aggregate is deeper nowadays, the mechanisms driving this process are still poorly understood. Among the factors involved, membranes should be taken into account. Indeed, convincing evidence suggests that membranes may influence protein folding, misfolding and aggregation. In fact, membrane lipid composition of different cellular types may attenuate or intensify the environmental pressure over protein folding equilibrium. In the present review the aim is to make an up-to-date analysis of the membrane influence on protein aggregation from a biophysical point of view in order to provide useful tools for researchers from other fields. In particular, we discuss how membranes can alter protein environment, e.g. increasing local protein concentration, lowering pH and dielectric constant, allowing accessibility to the hydrophobic milieu and promoting surface crowding, all of which will lead to protein aggregation. In addition, we review the role that specific lipids may exert on protein aggregation and finally we analyse the possible implication of membrane-related oxidative stress on amyloidogenesis.

Cell viability depends on the correct folding of the proteins involved in metabolism. Proteins are synthesized on the endoplasmic reticulum and must follow a pathway to a correct, metastable, tridimensional structure. Changes in structure or in environmental conditions can drive an instability of the folding conditions and produce non-active aggregates that in principle are proteolysed by the cellular mechanisms. However, these aggregates can be even more stable than the native proteins, escaping the cellular control. They can be classified as amorphous, if there is not a well-organized structural pattern, or ordered if a repetitive pattern is produced. These ordered structures, known as fibrils, are involved in many diseases. Infrared spectroscopy is a method of choice to study its formation because it is not affected by turbidity or the formation of high molecular weight aggregates. Moreover, in both cases, two bands characteristic of intermolecular - sheets allow the monitoring of the aggregate formation. In both cases, the appearance of these bands involves a nonreversible path in protein folding. It has been suggested that a difference in the ordered structures involves an increasing in band intensity. This change can be the origin in variations on the 2DCOS maps. The synchronous map gives an overall idea of the process involved. The asynchronous is more informative because reflects the kinetic changes produced. The outcome of both processes, amorphous or ordered is that 2DCOS can provide a further insight to the knowledge of the kinetic processes giving rise to aggregated structures. This outcome could consist on the order in which the different secondary structures are prone to form the aggregates.

The misfolding of proteins into a toxic conformation is proposed to be at the molecular foundation of a number of neurodegenerative disorders including Alzheimer's and Parkinson's diseases. Evidence that α-synuclein amyloidogenesis plays a causative role in the development of Parkinson's disease is furnished by a variety of genetic, neuropathological and biochemical studies. There is a major interest in understanding the structural and toxicity features of the various species populated along the aggregation pathway of this protein. The development of multidimensional Nuclear Magnetic Resonance (NMR) spectroscopy in liquid and solid state over the last decade has significantly increased the scope of molecules that are amenable for structural studies. The aim of this review is to provide a picture of how NMR tools were used in concert to decipher the structural and dynamic properties of the intrinsically disordered protein α- synuclein in its native, oligomeric, fibril and membrane-bound states. Understanding the structural and molecular basis behind the aggregation pathway of α-synuclein is key to advance in the design of a therapeutic strategy.

The misfolding and aggregation of amyloidogenic polypeptides are characteristics of many neurodegenerative syndromes including Alzheimer's and Parkinson's disease. There is a major interest in the availability of amyloid-specific probes that exhibit fluorescence properties for its use as reporters of protein aggregation in spectroscopy and microscopy methodologies. In this review we intend to provide an overview of novel fluorescence-based probes and procedures applied for addressing fundamental aspects of amyloid self-assembly in vitro and in vivo. We highlight the utilization in vitro of several small-molecule fluorescent probes as extrinsic and site-specific reporters of amyloid formation, including single-molecule determinations. Detection of amyloid self-assembly employing compounds such as JC-1, DCVJ, ANS derivatives and luminescent conjugated polymers, as well as site-specific probes such as pyrene and ESIPT is discussed. We further review novel fluorescent probes developed for the non-invasive optical imaging of protein aggregates in vivo, including BTA-1, Methoxy-X04, NIAD-4 and CRANAD-2. Availability of increasingly versatile amyloid-specific fluorescent probes is having a very positive impact in the drug discovery and diagnostics fields.

The 60's gave birth to the practical implementation of classical mechanics to unravel the dynamics and energetics of biomolecules. In the 70's the use of generalized force fields and more advanced integrative solutions to the microscopic understanding of nature (like hybrid QM/MM) were introduced. During the 80's, algorithms to obtain free energy values were further developed and in the 90's practical integration schemes of molecular mechanics force fields with other levels of detail (QM on one extreme and advances in implicit solvation on the other) were implemented in widely spread software. In the first decade of the XXIst century a considerable effort has been put in two seemingly discordant models for the simulation of biomolecules. On the one hand, extraordinary advances in computing technologies (both in terms of processor power and of new efficient parallel and distributed computing schemas) have allowed researchers to deal with bigger systems and longer simulations, reaching molecular processes including millions of particles or lying in the milisecond scale. On the other hand, the realization that the relevant answers to many biomolecular problems are not homogeneously distributed through the molecular structure, something already envisioned by the QM/MM pioneers more than three decades ago, has led researchers to find smart ways of putting different emphases on different ranges of the spatial or system time scale. In this context, e.g., molecular aggregation represents a paradigm for multiscalability, as molecular recognition can be understood with simple (semi-)macroscopic electrostatic terms when the two fragments are far apart, while the atomic interactions need to be considered in full detail upon close distances. In this manuscript the current status of the techniques that use multiple scale representations of biomolecules are reviewed, and the findings are synthesized in a modular schema that can be extensively used when studying aggregation processes. It is shown that a smart alternative to brute force and massive computation of uninteresting regions in the all atom potential energy surface is the consideration of a simplified reference potential, explored thoroughly in the relevant regions, combined with a free energy perturbation approach that transforms this simple representation to a full atom representation.

Alzheimer's disease (AD) represents the most common form of senile dementia and represents a tremendous health problem as the world population is aging. AD is characterized by the accumulation of amyloid β-peptide (Aβ) in the brain and the loss of cholinergic neurons in the basal forebrain. Accumulation of soluble and insoluble assemblies of Aβ in the brain is a crucial event in AD pathogenesis and the presence of amyloid plaques in the brain is required for definitive identification of AD. Yet, there is no correlation between amyloid plaques and the degree of dementia. In the past two decades researchers have devoted their effort to study and explain the mechanisms involved in the pathology of this devastating disease. Studies from different areas of the natural and medical sciences have provided important information towards the elucidation of some of the pathological processes that take place in AD. An aspect of crucial importance is the aggregation state of Aβ peptide and its role in neuropathology. Here, we discuss recent studies aimed at the identification of Aβ protein aggregates, the characterization of their toxic potential and the development of therapeutic strategies that target Aβ aggregation.

A number of neurodegenerative diseases, as Parkinson, prion, and Alzheimer's diseases, has been directly associated with altered conformations of certain peptides or proteins that assemble to form highly organized aggregates, also called amyloid fibers. Glycosaminoglycans have shown to play important roles on fibrils formation, stability and resistance to proteolysis. This manuscript reviews from basic concepts on the biochemistry and biology of glycosaminoglycans to their implications in neurodegeneration with particular emphasis in pathologic protein aggregation. Prion protein, Aβ42, Tau, and α-synuclein, are all proteins that can interact with glycosaminoglycans. We document here how these interactions may modify protein conformation, aggregation kinetics, and fibers stabilization with important consequences in disease. We also raise questions which answers may make advance the understanding of the implication of GAGs in neurodegeneration.